Towards the Development of an in Vitro 3D Co-Culture Cancer Model for the Design of Dendrimer-Anti-Cancer Drug Conjugates  

Towards the Development of an in vitro 3D
Co-Culture Cancer Model for the Design

of Dendrimer-Anti-cancer Drug Conjugates

 

Justin
Moore, Elizabeth Bielski, and Sandro R.P. da
Rocha*

School
of Pharmacy & Chemical and Life Science Engineering

Virginia
Commonwealth University Ð Richmond, VA 23298-0533

Two dimensional
(2D) monolayer cells cultured on flat substrates have been for decades an
important in vitro tool used to test
and understand numerous biomedical problems. Traditionally, these cell culture assays
have been a monolayer of cells cultured in
vitro. However, these 2D cultures have recently come under scrutiny for
their significant departure when compared to the in vivo tumor microenvironment they are designed to mimic. In 2D
monolayer cell cultures cell-to-cell/cell-to-matrix interactions, biomedical
gradients and signaling pathways are lost, tumor characteristics that significantly
affect the drug transport. The long-range of this project is to establish a 3D
in vitro model to test the ability of dendrimer-anti-cancer therapeutics to
treat solid tumors, and the impact of their chemistry in terms of its ability
to penetrate deep regions of the solid tumor environment. The first part of my
project is establishing a more relevant model of the tumor microenvironment.
This consisted of a transition from the traditional 2D cultures to a 3D model
that better represents the architecture of solid tumors. Furthermore, the
establishment of a co-culture system using cancer-associated stromal cells was
an appropriate subsequent step in to achieve a more relevant in vitro system to
test and understand the cytotoxicity profiles of the different systems. The
stromal cell type used were cancer-associated fibroblast (CAF) because of their
important role in creating an extracellular matrix which has been shown to
impact drug penetration. Optimization of the ratios of cancer/stromal cell
lines was required to see what initial ratio would result in a 70%/30% stromal/cancer
cell ratio, a ratio representative of the in vivo microenvironment. Briefly, at
a range of different ratios of 4T1 metastatic breast cancer cells and N1H 3T3 GFP
fibroblast cells were seeded in flat-bottom 96 well plates, which were coated
with agarose gel. The MCS were allowed to grow for 4 days then were
dissociated. Then, using Flow Cytometry the co-cultured spheroids were characterized.
It was found that the fibroblast and 4T1 cells are growing with similar growth kinetics
within the co-culture. Previously, using an MTT assay, we tested the
cytotoxicity of doxorubicin (DOX) on mono-culture MCS, and then compared these results
to a 2D model system assay.  The MTT
assay showed that much higher concentration of DOX are required to achieve 50%
of cell death (IC50%) in the 3D spheroid (ca. 13 fold).
Additionally, Dendrimer-Dox-conjugate had an increased toxicity profiles in
both system which may suggest that the dendrimer conjugation has more ability
to penetrate the spheroids and each cell in general. Next steps include
establishing a cytotoxicity protocol for the 3D co-culture system and how
manipulating (size, charge, and functional groups) impacts the Dendrimer-DOX
penetration.   

Corresponding author: srdarocha@vcu.edu

Acknowledgements: VCU for a Start-up Grant; NSF-DMR Grant # 1643770 and VCU IMSD - NIHR25GM090084 for a scholarship for JM.